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Influência da amidação do peptidoglicano na resistência à lisozima em Staphylococcus aureus
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Staphylococcus aureus é uma bactéria patogénica oportunista com elevada capacidade de adquirir
resistência a antibióticos, sendo uma das principais causas de infeções hospitalares, especialmente as
estirpes MRSA. A amidação do resíduo D-iso-glutamato do pentapéptido do peptidoglicano, pelo
complexo enzimático MurT-GatD, é uma modificação secundária que ocorre na parede celular desta
bactéria. Esta reação é essencial para o crescimento ótimo e para a expressão da resistência aos
antibióticos β-lactâmicos e à lisozima, muramidase à qual S. aureus é intrinsecamente resistente.
Contudo, estudos anteriores sugerem que existem outros determinantes genéticos, para além dos genes
murT e gatD, envolvidos no fenótipo de resistência à lisozima, de forma dependente da amidação do
peptidoglicano.
Deste modo, foi construída uma biblioteca de 494 mutantes no background genético de
COLpCadmurT-gatD, mutante condicional do operão murT-gatD da estirpe COL, com recurso ao
transposão Tn551, de modo a identificar determinantes genéticos envolvidos na resistência à lisozima.
A caracterização de 415 mutantes foi realizada através de ensaios de resistência à lisozima, permitindo
selecionar 273 mutantes em que a inserção do transposão resultou num fenótipo alterado de resistência
à lisozima do mutante COLpCadmurT-gatD, de forma dependente da expressão de murT-gatD. A
integridade do promotor induzível foi confirmada para 241 dos mutantes selecionados, por análise da
resistência aos antibióticos β-lactâmicos. O DNA genómico de 187 mutantes foi extraído, digerido com
uma enzima de restrição de corte frequente e o mapeamento da inserção do transposão foi obtido por
Southern blotting, com recurso a uma sonda específica para o Tn551. Dos 138 mutantes que
apresentaram apenas uma inserção do transposão, foram observados 31 padrões de hibridação
diferentes. Por sequenciação total do genoma foram identificados o gene miaA, que codifica para uma
tRNA transferase, e o gene SACOL0710, que codifica para uma proteína hipotética, que poderão ser
novos alvos para o desenvolvimento de estratégias antimicrobianas.
Staphylococcus aureus is an opportunistic pathogen with high capacity to acquire resistance to antibiotics, being one of the main causes of nosocomial infections, especially MRSA strains. Amidation of the D-iso-glutamate residue of the peptidoglycan pentapeptide by the MurT-GatD enzymatic complex is a secondary modification that occurs in the cell wall of this bacteria. This reaction is essential for optimal growth and for the expression of resistance to β-lactam antibiotics and lysozyme, a muramidase to which S. aureus is intrinsically resistant. However, previous studies showed that other genetic determinants, in addition to the murT and gatD genes, are involved in the lysozyme resistance phenotype, in a way that is dependent on peptidoglycan amidation. To identify these genetic determinants, a transposon mutant library with 494 mutants was constructed in the genetic background of COLpCadmurT-gatD, murT-gatD conditional mutant of strain COL, using the Tn551 transposon. The characterization of 415 mutants was performed by lysozyme resistance assays, allowing the selection of 273 mutants in which the transposon insertion resulted in an altered phenotype of lysozyme resistance of COLpCadmurT-gatD, in a mechanism that is dependent on the expression of murT-gatD. The integrity of the inducible promoter was confirmed for 241 of the selected mutants, through the analysis of resistance to β-lactam antibiotics. The genomic DNA of 187 mutants was extracted, digested with a high frequency restriction enzyme and the mapping of the transposon insertion was analyzed by Southern blotting using a Tn551-internal probe. Of the 138 mutants that showed a single transposon insertion, 31 different hybridization patterns were observed. Whole genome sequencing allowed the identification of the genes miaA (encoding a tRNA transferase) and SACOL0710 (hypothetical protein), which can ultimately be used as targets for the development of new antimicrobial strategies.
Staphylococcus aureus is an opportunistic pathogen with high capacity to acquire resistance to antibiotics, being one of the main causes of nosocomial infections, especially MRSA strains. Amidation of the D-iso-glutamate residue of the peptidoglycan pentapeptide by the MurT-GatD enzymatic complex is a secondary modification that occurs in the cell wall of this bacteria. This reaction is essential for optimal growth and for the expression of resistance to β-lactam antibiotics and lysozyme, a muramidase to which S. aureus is intrinsically resistant. However, previous studies showed that other genetic determinants, in addition to the murT and gatD genes, are involved in the lysozyme resistance phenotype, in a way that is dependent on peptidoglycan amidation. To identify these genetic determinants, a transposon mutant library with 494 mutants was constructed in the genetic background of COLpCadmurT-gatD, murT-gatD conditional mutant of strain COL, using the Tn551 transposon. The characterization of 415 mutants was performed by lysozyme resistance assays, allowing the selection of 273 mutants in which the transposon insertion resulted in an altered phenotype of lysozyme resistance of COLpCadmurT-gatD, in a mechanism that is dependent on the expression of murT-gatD. The integrity of the inducible promoter was confirmed for 241 of the selected mutants, through the analysis of resistance to β-lactam antibiotics. The genomic DNA of 187 mutants was extracted, digested with a high frequency restriction enzyme and the mapping of the transposon insertion was analyzed by Southern blotting using a Tn551-internal probe. Of the 138 mutants that showed a single transposon insertion, 31 different hybridization patterns were observed. Whole genome sequencing allowed the identification of the genes miaA (encoding a tRNA transferase) and SACOL0710 (hypothetical protein), which can ultimately be used as targets for the development of new antimicrobial strategies.
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Palavras-chave
Staphylococcus aureus amidação lisozima interação hospedeiro-microrganismo miaA SACOL0710